The information provided below is not admitted to be prior art to the present invention, but is provided solely to assist the understanding of the reader.
Systemic Lupus versus local Autoimmune Diseases. Lupus is the prototypical disease of a generalized autoimmune dysfunction and immune dysregulation. Many of the diseases considered to be autoimmune have manifestations of local immune dysfunction only, as in Diabetes, Thyroid disease, Multiple Sclerosis, Crohns disease, Colitis, Ankylosing spondylitis, Psoriasis, gastritis caused by Helicobacter pylori, and the central nervous system disorders Alzheimers, ALS, and Parkinsons disease. Peripheral nerve-neuropathic disorders, skin disorders and generalized arteriosclerosis, affecting the coronary, cerebral, and peripheral arteries all have now been shown to be caused by locally induced chronic inflammation. They all show much less generalized immune dysregulation than that which is seen in Lupus.
Systemic Lupus-Autoimmunity Tolerance Immune Dysregulation. Autoimmunity is considered to result and arise from the loss of Tolerance. Tolerance itself can be defined as the absence of an immune response to self and or other exogenous antigens. It can be ascribed to Innate, T and B cells and be manifested by failure of one or other of these individual cellular responses to the test antigens (which may be self or non self). It can also be due to loss of suppressor regulator T cells, both in number, and function. It can be central ie. arising from the thymus, or peripheral, involving peripheral T cells which fail to be anergized. Most available data on human Lupus (the prototype of a generalized autoimmune disease) and the equivalent mouse disease models, consider loss of anergy induction to be centered at the peripheral level. Suppressor T cell dysfunction has been associated with Lupus (Bresnihan and Jasin 1977; Horowitz, Borcherding et al. 1977; Morimoto 1978; Sagawa and Abdou 1978; Tardieu and Dupuy 1978; Kaufman and Bostwick 1979; Morimoto, Abe et al. 1979) (Abdou 1976, Krakower 1980) and both T cells and Monocytes have been shown to be important in this suppression. (Markenson, Lockshin et al. 1980) Furthermore anti T cell auto-antibodies and immune complexes have been implicated in loss of this T cell suppression, (Morimoto, Abe et al. 1979) which has been improved by, and following plasmapheresis. (Horwitz and Cousar 1975) When suppressor T cell function is lost there is an exaggerated B cell response to self antigens which results in a generalized autoimmunity as is seen in Lupus where antibodies are found to red cells, white cells, platelets, nucleic acids, nucleosomes, and other cellular components related to the apoptotic and necrotic cell death of T cells and Monocytes.
The immune system is essential for defending the organism against foreign invaders-bacteria, viruses, fungi and the like. It is generally assumed that the components of these offending agents or antigens are Non-Self, as distinct from self antigens such as dsDNA which has been shown to be critical in the etiology of Lupus and in particular the associated nephritis which arises from the deposition of immune complexes of dsDNA and anti DNA antibodies, in the kidneys of these patients. Most theories of Lupus etiology focus on genetic disturbances of T cell function (Tsokos 2003), and resultant T cell dysfunction. This T cell dysfunction or deficiency, in turn causes loss of T cell control of T cell dependent B cell function. The result is B cell clonal expansion and hyper-reactivity.
Another distinct and clearly different possibility which we here propose is that all self antigens preferentially induce central and peripheral Tolerance or immuno-suppression by activation of specific suppressor T cells and monocytes. Loss of this suppressor regulator T cell function which is central to this theory would predictably give rise to loss of self antigen Tolerance and result in antibodies and T cell responses to self antigens, and result in auto-antibodies, and T cell inflammation in organs, and auto-immune diseases. It is therefore responsible for the disturbed homeostasis and immune dys-regulation which follows, which is central to Lupus pathogenesis, and to other allergic and autoimmune disorders. (Von Herrath et al. Nat Rev Immunol 223-232 2003).
Arthritis. This is caused by over 100 different diseases or disorders, and it affects over 70 million Americans. Rheumatoid Arthritis, Systemic Lupus Erythematosus, Scleroderma, Polymyositis, Mixed Connective Tissue Disease, Vasculitis affecting both large and small vessels, are all serious, potentially lethal, disabling diseases with high morbidity, high mortality, high dollar cost to the individual, the community, and the nation. They are all classified as Connective Tissue Disorders, or Disorders of persistent antigenic stimulation and result in Immune Dys-Regulation. Recent epidemiology data suggest that together they affect more than 30% of the US population, at a cost of over $12,000 per patient annually. All of these are considered to be of unknown cause or idiopathic, in nature. They are thus considered to be non infectious, and autoimmune, in that no identifiable organism has been identified as the primary cause of these illnesses.
Arterial-Degenerative Diseases.
Degenerative and inflammatory disorders involving the Coronary, Cerebral, and Peripheral arteries account for the greatest number of deaths annually in developed countries, and with increasing frequency the developing countries are following a similar pattern. Increasing evidence has been cited that inflammation in addition to high cholesterol, lack of diet and exercise, obesity and diabetes, play significant roles in causing this arterial degeneration. Thus cardiac C Reactive Protein CRP, has been identified as the highest risk factor for an acute coronary event in humans. This protein is produced by the liver in response to IL-6, and CRP is thus an inexpensive measure of IL-6. The annual cost in medical interventions, hospital costs, nursing home costs, insurance costs and disability costs have been estimated to be in the billions of dollars annually in the US alone. The precise cause of this inflammation is not yet clear but infections such as Helicobacter, Chlamydia, CMV, and others alone or by inducing heat shock proteins have been suggested. The latter have been shown to become auto antigens in CAD and Carotid artery disease. While the precise trigger of this inflammation is still to be determined, the presence of inflammation at the endothelial lining of the arteries is no longer disputed. (Ross R., N.E.J. Med. 1999 340:115-226).
The use of Statin drugs, and awareness of the role of diet and exercise, have for the first time begun to cause a reduction in the frequency of deaths and disability form these diseases, previously lethal at an early age. It has recently been shown that statin drugs, invented for their use as cholesterol lowering agents, are anti-inflammatory. They have even been shown to be efficacious in the treatment of Multiple Sclerosis, an inflammatory disorder of the brain caused by persistent immune activation. Statins may therefore influence disease patterns and outcomes by reducing inflammation as well as by lowering cholesterol levels.
Alzheimers disease. This is the single most important cause of dementia in developed countries where and when life expectancy has been increased. Recent statistics on this disease suggest that 1 in 4 or even more over 80 years of age will succumb to this disease. Over 20 studies have now shown that anti-inflammatory non steroidal aspirin like drugs, alter or suppress the progress of this disease. Amyloid fibrils, formed as result of cleavage of Amyloid Precursor Protein APP, by enzymatic cell membrane secretases, are formed from the Aβ fragments produced by this cleavage. They are deposited in the brain at the synaptic clefts where acetylcholine ACH, and butyryl choline BCH, are produced and which are responsible for the transmission of the nerve impulses. Atrophy and loss of the neurons due to these amyloid proteins causing neural tangles, and Tau proteins causing neurofillament destruction, together with the major reductions of ACH and BCH levels on brain biopsies are considered the pathological and biological hallmarks of Alzheimers disease. Proinflammatory cytokines when measured in the brain tissues as messenger RNA, predate the finding of Aβ in the brain. Chronic inflammation is thus an important mechanism in its causation.
The brain is a privileged site and evolved prior to the development of T cells and B cells. These cells should consequently never be seen within this privileged site, the brain, unless there is inflammation in it, or in the adjacent meninges or blood vessels. The more primitive innate immune cell system is represented here however by microglia and astroglia. Cells of the innate system in responding to antigens or triggers are known to produce the pro-inflammatory cytokines TNF-α, IL-1, and IL-6. All three cytokines are found in and around the synaptic clefts at the site of deposition of the amyloid fibrils. Thus the finding of both amyloid fibrils and these Cytokines are evidence for persistent immune activation at the sites of neuronal damage, nerve cell atrophy or drop out, and ACH and BCH reductions. (McGeer & McGeer Science SAGE KE 2004 27: 29) Thus persistence of immune activation in response to as of yet some unknown trigger is the most likely cause of Alzheimers disease. No single method to abort the disease, or the deposition of amyloid and Tau proteins is currently available. All treatments are for symptoms only, where increase in ACH and BCH levels improve function temporarily.
Amyloidosis. This is a systematic disease caused by deposition Amyloid fibrils in organs and tissues such as spleen, liver, kidney and heart, in mammals including humans by persistent activation of the immune system. Diseases such as Tuberculosis, Osteomyelitis, and Leprosy, all of which are due to persistent infections, are frequently associated with systemic amyloidosis. This disease is uniformly fatal as no therapy has been identified to terminate the production of the amyloid proteins in the affected organs. Isolated organ death, or organ failure, is the usual cause of death in this disease. Organ grafting of kidney, heart, lung, liver, or bone marrow, is used, but the immunosuppressive regimens used to inhibit the GvH and HvG response to grafting may aggravate and accelerate the systemic amyloidogenic process, and thereby negate the effectiveness of the transplants. An effective method to reduce the chronic inflammation and immune activation is critically needed to treat this lethal disorder.
Organ Grafts and Disease. Organ grafting of kidney, heart, lung, pancreas, liver, bone marrow, stem cell, and bone marrow, are standard practice in the medical arsenal for responding to isolated organ failure, with a view to improving the quality and quantity of life, of the recipients. Organs other than autologous stem cell and bone marrow which are transplanted are obtained from unrelated donors, (allogeneic). As such there are genetic differences between the donors and the recipients. The resulting immune reactions which develop emanate from attempts by the recipient or the donor immune cells to immunologically reject the graft or the host cells. The former is termed host versus graft (HvG), and the latter graft versus host rejection (GvH), and disease. Allogeneic organ grafts and the HvG immune responses have been successfully controlled by the use of immune-suppressants. Because stem cell and bone marrow grafts include pluripotent immune cell progenitors there is a major need to alter the frequency and severity of the GvH disease resulting from allogeneic stem cell and bone marrow transplants. Successful intervention to prevent this GvH disease by acquiring Tolerance (Billingham Brent and Medawar Nature 1953 172; 603-606), would alter the underlying approaches to the treatment of Bone Marrow failure, spontaneously developing, or induced, and in the treatment of cancers of multiple types, and resistant autoimmune diseases, where bone marrow failure is the unwanted outcome of aggressive chemotherapy to alter immunity and kill the cancer cells.
Autoimmunity and Autoimmune Disorders and Diseases. Autoimmunity is defined as persistent and progressive immune reactions to non infectious self antigens, as distinct from infectious non self antigens from bacterial, viral, fungal, or parasitic organisms which invade and persist within mammals and humans. (Janeway 1992, & 1999).
The development of the newer biologics, in the form of monoclonal antibodies against the pro-inflammatory cytokines IL-1, TNFα, IL-6, and IL-1 receptor, and their use in Rheumatoid Arthritis, Psoriasis, Crohns Disease, Colitis, and Ankylosing Spondylitis has resulted in major benefits characterized as clinical remissions or reductions in inflammation when monitored by ACR 20, ACR 50, and ACR 70 outcome responses; reduction in bony erosions on X Ray analysis, and in the prevention or reduction in deformities and disability. These improved outcomes have been major treatment advances allowing patients to reduce or eliminate the use of corticosteroids and some chemotherapy drugs which had previously been the standard of care for these diseases. These major advances have also revolutionized our understanding of the mechanism of these diseases. This excitement has been somewhat tempered by the development of bacterial infections, fungal and TB infections, in some patients, and in small numbers of reported deaths from cancer, both lymphoid and non lymphoid malignancies. These latter responses at this time are not statistically significant however. Nevertheless they are of concern in that they may increase in frequency as the duration of the use of these biologics increases. Gradual increase in dose requirements in order to maintain remission (Remicade), the development of antibodies against the biologic (Remicade), skin reactions (Enbrel and Kineret), the development of autoantibodies, vasculitis, and illnesses as are seen frequently in Lupus, are further complications which may limit the use of these biologics in the future.
Even with the use of all of these treatments individually, and combined, clinical response failures do exist, and have been cited as high as 50%. (Firestein 2003) Recourse then to plasmapheresis, and immunoadsorbent columns to remove inflammatory molecules, and to autologous and allogeneic bone marrow and or stem cell transplants, or to monoclonal antibodies against B cells and or the co-stimulatory molecules are the last resort for maintaining a pain free quality of life in this small group of response failures. The need for newer agents and treatments with out such complications or side effects is therefore apparent.
Background of CTdsDNA as an Immune Activator/Suppressor. Several hundred patents reviewed included the use of DNA as antigen vectors, immune enhancers, or adjuvants for vaccines. None of the patents already approved or pending identified CT DNA as an inducer of Suppressor/Regulator T cells, which then have the ability to suppress T cell and B cell stimulation and blastogenesis, to antigens, mitogens, and alloantigens.
Zeuner et al Arth &Rheum 2003, identified both immunostimualtory and immune suppressive oligodeoxynucleotides (ODNs) in mice. They suggested the use of suppressive ODNs may be useful in the prevention and treatment of proinflammatory diseases.
Zhao et al Antisense Nuceic Acid Drug Dev 2000 identified ODNs with CpG motifs as both neutral non stimulating and stimulatory introducing the idea that all CpG motifs were not stimulatory.
Mammone et al. Skin Pharmacol Appl Skin Physiol 2002. identified that UVL light induces damage to skin keratinocytes. They found that the trigger for repair was damaged DNA itself. They found that fragmented CT DNA irradiated and damaged with UVC light, caused a protective effect on the cultured keratinocytes. It caused a suppression of DNA synthesis in vitro thereby allowing repair. This effect was also dose dependent. The mechanism for this suppression was considered to be the p53 suppressor gene activation.
Yasuda et al Biochem Biphys Res Commun 2002 identified that naked CpG induced TNFα secretion from macrophages in vitro but DNA without CpG did not.
Wu et al PNAS 2002. Found that nonobese diabetic mice (NOD) have a deficiency of CD4 CD25 T cells in thymus and spleen. They suggest that a deficiency of Treg cells is responsible for diabetes induction and that as anti TNF neonatally administered prevents the disease, TNF in the thymus may have a role in disease induction. (IL-6 was not measured however in the study).
Bresnihan, B. and H. E. Jasin (1977). “Suppressor function of peripheral blood mononuclear cells in normal individuals and in patients with systemic lupus erythematosus.” J Clin Invest 59(1): 106-16. Normal peripheral blood mononuclear cells demonstrated increased DNA synthesis and secretion of newly synthesized protein when suboptimal concentrations of Concanavalin A (Con A) were added to the cultures after 24-h incubation in vitro. Cells stimulated by Con A, 1 mug/ml, after 24-h incubation demonstrated 3.0 times more tritiated thymidine incorporation, and 4.4 times more 14C-amino acid incorporation into newly synthesized secreted protein, than cells stimulated at 0 h (P less than 0.001). The acquisition of increased responsiveness was not abrogated by washing and resuspending the cells in fresh medium. Since the increased responsiveness could be inhibited by the addition to the cultures of small numbers of cells previously activated by Con A it is suggested that the enhanced reactivity acquired in culture represents the loss of a subpopulation of suppressor cells that modulate the T-lymphocyte response. Cells from nine patients with active, untreated systemic lupus erythematosus demonstrated normal responses to optimal concentrations of Con A added at 0 h, but an impaired response to Con A, 1 mug/ml. When these cells were incubated for 24 h, a significant increased response to Con A was not observed. This observation suggests that patients with active SLE lack circulating suppressor cells. When seven SLE patients were again studied after corticosteroid therapy had led to clinical improvement, the response to Con A, 1 mug/ml, added after 24-h incubation was similar to that observed in normal controls, suggesting that suppressor function in SLE returns as disease activity declines.
(Aldo-Benson 1989) found that B cell clones from both autoimmune disease (AID) prone and non AID prone mice could be maintained in culture and that added dsDNA induced T cells to the cultures suppressed the B cell clones in the non AID prone but not in the AID prone.
(Ochi, 1983 #258) found that procaine amide enhanced autoantibody production and AID in prone mice by altering suppressor T cell induction. (This drug is now known to demethylate CpGs, which may be the mechanism).
(Newman, 1979 #218) showed a lack of Ts suppressor T cells in Lupus. (Miller, 1989 #437) identified a thymic factor TsIF which was produced from thymic epithelial cells co-cultured with T cells and which upon injection could suppress Lupus and Rheumatoid arthritis inflammation in AID prone mice. The data suggests a soluble factor from thymus can induce T cell suppression.
Horowitz, S., W. Borcherding, et al. (1977). “Induction of suppressor T cells in systemic lupus erythematosus by thymosin and cultured thymic epithelium.” Science 197(4307): 999-1001.
(Blank, 1991 #192) showed dsDNA specificity to the induction of Ts cells in suppression of B cell autoantibody production.
(Borel, 1980 #177) was the first to show nucleic acid specific induced B cell suppression and considered it a potential method of treatment for AID.
(Chen, 2001 #935) in an effort to understand the differences in DNA sources as immune activators/suppressors found that E coli (EC) DNA was an immune inducer while calf thymus DNA (CT DNA) was an immuno-suppressor. The former contains CpG motifs which are unmethylated, and which are not found in CT DNA. The mechanism for EC stimulation was different from but synergistic with Endotoxin. The mechanism for immunosuppression was considered to be due to inhibition of the EC induced signaling by NFkappa-B and AP-1. (It is now known that EC CpG motifs activate TLR 9 (O'Neill 2003) and endotoxin TLR 4, and the fact that dsCTDNA suppresses EC but not endotoxin suggests the silencing of activation may be through TLR 9 or its adapter proteins, or transcription factors).
(Schwartz, 1997 #879) showed that EC DNA with CpG motifs activate inflammation in the lungs also.
(Segura-Pacheco, 2003 #1) in an interesting study showed that the drug hydralazine—a known inducer of Lupus in man de-methylates DNA. Because activation of suppressor T cells is known to be associated with certain cancers, they suggested that the use of this drug may alter the suppressor T cell state found in cancer, by re-initiating immune activation by DNA de-methylation.
Circulating DNA has been reported in the plasma of patients with different cancers, aging, pregnancy and other states. It may therefore play a significant role in the immunosuppression associated with both pregnancy and certain cancers. Suggestions for the use of immunostimulatory DNA with unmethylated CpG motifs to upregulate DCs, CD 4 and CD 8 to kill cancer cells have accordingly been entertained.
Other objects and advantages will become apparent from the following disclosure.